The present invention is concerned with a process for the detection of substances with hydrolase activity and with an agent suitable for carrying out the process, as well as with new hydrolase substrates and processes for the preparation thereof.
Hydrolases are enzymes which have recently achieved great importance. On the one hand, they play an important role in the metabolism of plants and animals and also of humans. If the concentration of a hydrolase in one of these biological systems deviates from the concentration normally present therein, then this can be the cause of a disease. Therefore, it is a task of clinical diagnosis, in the case of the presence of a disease, to ascertain, by determination of the concentration of a hydrolase in body fluids, possible deviations from the normal value. This preferably takes place via the determination of the hydrolase activity by means of an indicator reaction. For this purpose, the sample to be investigated is mixed with a substrate for the hydrolase in question. The amount of product formed from the substrate is a measure for the hydrolase concentration present.
On the other hand, hydrolases are used ever more frequently as enzymes for labelling immunologically active compounds. .beta.-Galactosidase is hereby especially widely used for the labelling of, for example, antibodies in immunological tests (see Annals of Clinical Biochemistry, 16, 221/1979). Tests of this kind serve to determine the content of an immunologically active analyte in a sample. They are so constructed that the concentration of the analyte is determined via an appropriate immune component which carries covalently bound .beta.-galactosidase as label. The test is so constructed that the concentration of the immune partner is directly dependent upon the concentration of the analyte to be determined. The concentration of the labelled immune component is also made visible via an indicator reaction, in which the immune component labelled with B-galactosidase is reacted with a substrate for B-galactosidase.
The amount of product formed is proportional to the concentration of the immune component. By comparison with the values of a calibration curve, which is produced with the help of samples with known analyte concentration, an unknown analyte concentration in a sample can be determined.
Hydrolases are also used as enzymes for labelling nucleic acids in processes for the detection of nucleic acids. Such a process which uses .beta.-galactosidase as enzyme label is described in Federal Republic of Germany Patent Specification No. 29 15 082. Here, too, the amount of label is determined in an indicator reaction.
Furthermore, hydrolases are used as reagents in research. Here, too, it is important to be able to determine the concentration of the enzymes exactly and quickly. In general, for this purpose, there are used methods which are similar to those used in clinical diagnosis.
The substrate used is generally a chemical compound which is soluble in the sample liquid. In the case of the reaction with the hydrolase, a product is formed which differs in one of its characteristics, for example a characteristic light absorption, a light emission (fluorescence) or the like, from the substrate and can thereby be determined.
In the case of test processes on immunologically active substances, a differentiation is made between two technical embodiments. In the case of a frequently used embodiment, the detection reaction-takes place in a cuvette such as is generally used for photometric investigations. The evaluation of the indicator reaction is then carried out by measurement of the absorption, emission or radio-activity in an appropriate apparatus.
In the case of another embodiment, the reactions take place in one or more fleece or films which are parts of a test carrier, the necessary reagents being applied to these fleece or films. The amount of product which is liberated by the hydrolase activity from the hydrolase substrate during the indicator reaction can then be determined directly in such a fleece or film. An advantage of this embodiment is that it is possible to work with only a single solution, the liquid sample, which considerably simplifies the carrying out of the process. The amount of product formed can be detected especially simply by measurement of the light absorption at a particular wavelength.
Substrates suitable for this purpose include the phenyl-sulphophthaleinyl-.beta.-D-galactosides and derivatives thereof, which are described in European Patent Specification No. 01 46 866, from which phenolsulphophthalein derivatives are liberated by reaction with .beta.-D-galactosidase. Whereas the substrate has a yellow colour, the product is red coloured. Thus, during the indicator reaction, a gradual colour change from yellow via orange to red takes place. The colour change can only be assessed very inaccurately with the naked eye so that an appropriate photometer must be used for the measurement.
The resorufin-.beta.-D-glycosides described in European Patent Specification No. 0 156 347 are also substrates in which case the reaction with a glycosidase causes a colour change from yellow to red to take place. It has been ascertained that in the transitional region between yellow and red, especially in the case of low .beta.-glycosidase activities, the visual evaluation leads to subjective results, for which reason apparatus are here also used for the evaluation. However, these apparatus are relatively complicated, and, therefore, expensive. The resorufin glycosides are not very suitable as substrates for use in test carriers since resorufin formed from them bleeds out.
Hydrolase substrates such as, for example methylumbelliferone galactoside, in which hydrolysis causes only a change of the fluoresence properties, are also unsuitable for a visual evaluation. Methylumbelliferone galactoside also gives rise to a substance which easily bleeds out of the test strips.
Furthermore, processes for the detection of substances with hydrolase activity have been suggested in which uncoloured hydrolase substrates are used which, in the case of reaction with a hydrolase, are converted into coloured products. Consequently, in the case of these detection reactions, the formation of a colour is to be assessed rather than a colour change. This can take place in a simple way by comparison of the colour intensity with a colour shade of a colour scale. Even colour-blind persons can evaluate such a test.
5-Bromo-4-chloro-3-indolyl-.beta.-galactoside is such a substrate. This substrate forms after cleavage and oxidation a product which does not bleed out strongly from test carriers. However, the substrate itself has a low water-solubility and is cleaved only slowly by .beta.-galactosidase. Therefore, the resultant colour formation is not very intensive. A test constructed thereon is thus not suitable for a rapid visual evaluation.
In Biochem. Z., 333, 209/1960, compounds are suggested as substrates, the hydrolysis of which liberates phenols. However, these readily bleed out of test carriers. In the case of nitrophemol, a more or less intensive yellow coloration develops during the indicator reaction. In the case of this wavelength, the visual evaluation in the range of small concentrations is also very unfavourable.
For improving visibility, in an additional reaction the liberated phenols are oxidatively coupled with aminoantipyrine or methylbenzothiazolinone hydrazone (Anal. Biochem., 40, 281/1971) or with diazonium salts to give coloured azo materials (see, for example, Histochemie, 37, 89/1973). Some of these coloured materials are admittedly suitable for use in test carriers since they do not bleed out so easily, but the use of this coupling reaction is disadvantageous for other reasons. Thus, for the carrying out of the test on a fleece or film, all the reagents necessary for this additional reaction must also be applied to a fleece or incorporated into a film. The large number of reagents, for example coupling components, complicates the carrying out of the test and gives rise to numerous disadvantages and difficulties. Thus, in individual cases, care must be taken that the reagents not react prematurely with one another or non-specifically with other components of the sample solution. Thus, aminoantipyrine or methylbenzothiazolinone hydrazone also react with components present, for example, in a urine sample to be investigated and thus falsify the measurement. Other coupling components, such as diazonium salts, impair the storage stability. In the case of the choice of the reagents, it is also to be taken into account that they do not themselves already possess an inherent colour.
Bleeding out phenomena, which occur on test carriers in the case of some of the dyes formed, give, for example, the result that the colour intensity is not the same at all places on the fleece or film. This gives rise to disadvantages in the evaluation since, for example, the measurement values are falsified.
Therefore, there is a need for a process in which the described disadvantages are avoided and with the help of which substances with hydrolase activity can also be detected on test carriers in a simpler, quicker and more dependable way.
It is an object of the present invention to satisfy this need.